Fibrinogen is the plasma protein responsible for blood clot formation. Normal fibrinogen is a complex of 2 each of 3 chains (α, β and γ) (1-6). A variant of α fibrinogen (fib340) with an extended α chain known as αE fibrinogen (fib420) that constitutes about 1% of the total fibrinogen in adult humans has more recently been discovered but its unique function is not yet clear (7-10). Thus, the four types of fibrinogen chains, α, β, γ and αE, contain 610, 483, 411 and 866 amino acids, respectively (the numbering based on the Gene-bank database, accessible at ncbi.nlm.nih.gov).
Fibrinogen is not immunogenic within the same species, as attested by the use of pooled fibrin glue for clinical applications. Besides its hemostatic activity, it has been previously demonstrated that fibrin(ogen) elicits cell attachment (haptotactic) and migratory (chemotactic) responses with different cell types including mouse and human fibroblasts (MF and HF), bovine aortic endothelial (BAEC) and smooth muscle cells (SMC) (11, 12).
The carboxy terminal sequences, i.e., the C-terminal 30-40 amino acids of the fibrinogen chains, are highly conserved between different species (13, 14). With the exception of the γ-chain C terminus (11, 12), they have not been shown to relate to any hemostatic function of fibrinogen. A voluminous literature exists which describes the binding of fibrinogen (γ400-411) to platelets through the GPIIb/IIIa receptor and the aggregation activity of the new amino Bβ15-42 terminus that is exposed after release of fibrinopeptide B.
Fibrinogen fragment E was reported to exhibit angiogenic properties and to inhibit endothelial cell migration in a Boyden chamber chemotactic assay (19). The larger fragment D was reported to cause detachment of cultured endothelial cells from the extracellular matrix (ECM) substratum in a concentration and time dependent process (20).
Isolated constituent chains of fibrinogen (Aα1, Aα2 and Bβ) released upon activation of fibrinogen by thronibin were observed to stimulate fibroblast proliferation by 23-31% above controls, whereas isolated γ chain had no effect (22). Human polymorphonuclear leukocytes (PMN) were shown to bind to fibrin(ogen) coated surfaces via a type 3 (CD11b/CD18) complement receptor homologous to the GPIIb/IIIa receptor through a decamer of the γ chain carboxy terminus (LGGAKQAGDV, SEQ ID NO:18). Vasoactive peptides were identified corresponding to residues 43-47 of the Bβ chain and 220-230 of the Aα chain (21).
The biological activities of a few other fibrinogen breakdown products have been investigated, but the cellular activity seemed to be widely variable (23).
Functional peptide sequences previously have been disclosed on the γ-chain, including sites involved in platelet binding (γ 400-411), leukocyte adhesion (γ 396-411), factor XIII-crosslinking sites (γ 398, γ 407), a polymerization region (γ 374-395), and fibroblast adhesion region (γ 374-394). Thus, fibrinogen interactions with platelets and cells have been documented by a number of workers.
It has previously been disclosed by the present inventors (WO99/61041) that certain cell attachment effects of the intact fibrin(ogen) could be ascribed to small sequences at the carboxy termini of all the fibrinogen chains. Synthetic peptide fragments of the last 19-21 amino acids of carboxy termini of the α, β, and γ chains of normal fibrinogen and of the αE chain (peptides termed Cα, Cβ, Cγ and CαE respectively), were tested. Only Cβ and CαE sequences induced significant haptotactic responses from various cultured cell types, mostly of mesenchymal origin, such as HF, BAEC and SMC, whereas the Cα and Cγ peptides did not exhibit significant haptotactic (cell attachment) activity. The active peptide Cβ was shown to be rapidly taken up by the cells in a non-saturatable manner. None of the disclosed peptides affected the rate of cell proliferation.
The identification of new haptotactic epitopes would have a number of applications, enabling more specific intervention in the wound healing process and the development of novel therapeutic compositions or devices. Furthermore, novel diagnostic tests to monitor cellular haptotactic responses could potentially be developed. Such peptides may have the ability to elicit haptotactic responses from cells, with no need to utilize the whole fibrinogen molecule and its attendant safety and regulatory issues.
Thus, there is a recognized need for, and it would be highly advantageous, to have peptides with specifically determined cellular effects, such as chemotactic or haptotactic properties, which do not require the presence of the entirety of the fibrin(ogen), or the entirety of other proteins containing a homologous sequence.